The Caliciviridae are a family of positive-strand RNA viruses and consist of four genera designated: (1) Norovirus (with species Norwalk virus) (2) Sapovirus (with species Sapporo virus); (3) Vesivirus (with species, feline calicivirus and vesicular exanthema of swine virus); and (4) Lagovirus (with species rabbit hemorrhagic disease virus and European brown hare syndrome virus). Norwalk virus (NV) is the prototype strain for the genus Norovirus and was discovered by LID researchers in 1972. The Noroviruses are the major cause of nonbacterial epidemic gastroenteritis that occurs in family, school, institutional, or community-wide outbreaks, affecting all age groups. The Noroviruses are genetically diverse and cannot be grown in cell culture, which has been a continuing research obstacle. A major goal of this laboratory is the development of control strategies for the caliciviruses (predominantly the noroviruses) associated with gastroenteritis. In order to accomplish this goal, basic knowledge of the epidemiology, immunology, and replication strategies of these viruses is needed. This year, we continued our comparative studies of representative viruses from three distinct Caliciviridae genera- Norovirus, Sapovirus, and Vesivirus. Two viruses, feline calicivirus (Vesivirus) and porcine enteric calicivirus (Sapovirus) grow in cell culture, and we have developed both systems further to study replication in cells. We established a cDNA clone-based reverse genetics system for porcine enteric calicivirus that is the first such system for an enteric calicivirus pathogen. The recovery of porcine enteric calicivirus from infectious RNA transcribed from the full-length cDNA clone required the presence of intestinal content (IC) fluid from an uninfected pig in the cell culture medium (IC is required also for the growth of the wild type virus). Progress was made in isolating active fractions from the IC by size exclusion chromatography that support virus growth in cell culture, and the identification of this factor could be important in efforts to grow the noroviruses. In addition, we showed for the first time that apoptosis of virus-infected cells is triggered by active feline calicivirus replication, and gained insight into the specific viral proteins responsible for this process. This information could facilitate the development of efficient calicivirus-based expression systems or replicons. We also completed a study that mapped the second tyrosine of the VPg protein of feline calicivirus as the likely amino acid that mediates linkage of this small protein to the viral RNA genome. Since norovirus strains such as Norwalk virus and MD145-12, cannot yet be grown in cell culture, we focused on study of their nonstructural proteins in in vitro systems. We discovered that prolonged incubation of coupled transcription/translation reactions allowed proteolytic processing of the nonstructural polyprotein into both precursors and final products that were analogous to those of the caliciviruses above that grow in cell culture. The identification of these precursors has established a foundation for future studies of protein structure and function that may facilitate the development of antiviral drugs. We initiated two new collaborations this year. One collaboration is with Dr. Herbert (Skip) Virgin at Washington University in the characterization of a new murine norovirus, and the other is with Dr. Richard Lloyd at Baylor University relating to the interaction of calicivirus nonstructural proteins with the cellular translation machinery.